Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology.

BACKGROUND

Rotavirus is the representative cause of severe acute gastroenteritis in young children. A characteristic feature of rotavirus is low infectious dose and robustness of the virion, suggesting sanitation and hygiene will have little impact. Thus, development of a vaccine should be given priority. Efficient capture of infectious viruses is an important step in generating a vaccine. Previously, antibody-integrated magnetic nanobeads (MNBs) have been developed for virus capture. This study examines the applicability of this method for infectious rotavirus recovery and enrichment.

MATERIALS AND METHODS

Graphite-encapsulated MNBs were treated with radio frequency (RF) excited Ar/NH gas mixture plasma to introduce amino groups onto their surfaces. Rotavirus viral protein 7 (VP7) antibody was attached to the surface of MNBs via these amino groups using a coupling agent, -succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The antibody-integrated MNBs were incubated with rotavirus-infected cell lysate and then separated from the supernatant by applying a magnetic field. After thorough washing, rotavirus was recovered and enrichment analysis done by polymerase chain reaction (PCR), immunochromatography, and an infection analysis using MA104 cells.

RESULTS AND DISCUSSION

Immunochromatography and PCR indicate that anti-rotavirus antibody-integrated MNPs efficiently capture rotavirus with the capsid protein and viral RNA. The estimated recovery rate was 80.2% by PCR and 90.0% by infection analysis, while the concentrating factor was 7.9-fold by PCR and 6.7-fold by infection analysis. In addition, the absence of non-specific binding to the antibody-integrated MNPs was confirmed using anti-dengue virus antibody-integrated MNBs as a negative control.

CONCLUSION

These results suggest that this capture procedure is a useful tool for recovery and enrichment of infectious rotavirus. Moreover, when combined with a suitable virus assay this capture procedure can increase the sensitivity of rotavirus detection. Therefore, this capture method is a valuable tool for generating vaccines as well as for developing sensitive detection systems for viruses.